Conductive Hydrogel and Integrated Bio-Photovoltaic System and Preparation Method Thereof

Abstract

The present invention belongs to the field of bio-photovoltaics, and particularly relates to an integrated bio-photovoltaic system based on conductive hydrogels. The present invention discloses a conductive hydrogel, which is composed of a hydrogel and a conductive material; the hydrogel includes a gelatin-carrageenan composite hydrogel, a sodium alginate-polyvinyl alcohol composite hydrogel, a sodium alginate-polyethylene glycol composite hydrogel, and a sodium alginate-carboxymethyl chitosan composite hydrogel; the conductive material includes polypyrrole, polyaniline, carboxylated multi-walled carbon nanotubes, amino-functionalized multi-walled carbon nanotubes, and conductive carbon black. The present invention also discloses an integrated bio-photovoltaic system composed of a conductive hydrogel and a synthetic microbiome. The advantages of the present invention are that the conductive hydrogel creates an anaerobic environment and a conductive network for heterotrophic microorganisms, solves the problem of "oxygen robbing electrons" in synthetic microbiome bio-photovoltaics, and enables photosynthetic microorganisms and heterotrophic microorganisms to generate electricity jointly in the same time and space.

Description

Technical Field

[0001] The present invention belongs to the field of biophotovoltaics, and particularly relates to an integrated biophotovoltaic system based on conductive hydrogels. Background Art

[0002] Biophotovoltaics is a bioelectrochemical technology that uses photosynthetic microorganisms as optoelectronic conversion materials to convert solar energy into electrical energy. As a transformative solar power generation technology, biophotovoltaics has the following advantages: (1) Biophotovoltaics uses the most abundant and cheapest energy resources and electron resources on earth, namely solar energy and water, for power generation; (2) Photosynthetic microbial materials are renewable and pollution-free; (3) Photosynthetic microorganisms can fix carbon dioxide during growth, having the function of carbon dioxide emission reduction.

[0003] Due to the relatively weak transmembrane electron transfer ability of photosynthetic microorganisms themselves, the output power of a single-bacterium biophotovoltaic composed of a single photosynthetic microorganism is relatively low. However, the use of a synthetic microbial consortium (photosynthetic microorganisms - energy carriers - electrogenic microorganisms) with directed electron flow can significantly improve the output power of biophotovoltaics (Zhu H., et al, Development of a longevous two-species biophotovoltaics with constrained electron flow. Nature Communications. 2019, 10, 4282). In the synthetic microbial consortium, photosynthetic microorganisms are responsible for capturing light energy and storing the energy into energy carriers (photosynthetic charging), and electrogenic microorganisms oxidize the energy carriers under anaerobic conditions and transfer electrons to extracellular electrodes to generate current (anaerobic power generation). The synthetic microbial consortium biophotovoltaic neither depends on the electroactivity of photosynthetic microorganisms themselves nor on exogenous electron carriers, and thus can perform optoelectronic conversion efficiently and stably.

[0004] In the synthetic microbial consortium biophotovoltaics, photosynthetic microorganisms produce a large amount of oxygen during photosynthesis, while electrogenic microorganisms (or heterotrophic microorganisms) preferentially perform aerobic respiration under aerobic conditions, thus dissipating all the energy. This process is called "oxygen stealing electrons". To overcome this problem, previous strategies were to perform photosynthetic charging and anaerobic power generation in two stages to avoid oxygen interference. However, the two-stage operation mode increases the complexity of the system and is not conducive to the scaling-up of the biophotovoltaic system.

[0005] The present invention completely solves the problem of "oxygen stealing electrons". By developing a biocompatible conductive hydrogel as an oxygen isolation medium, the entire synthetic microbial consortium is assembled into an integrated system, enabling multiple microorganisms to cooperate in power generation in the same space. This integrated biophotovoltaic system is very conducive to array scaling-up. Summary of the Invention

[0006] The object of the present invention is to solve the problem of "oxygen robbing electrons" in synthetic microbiome bio-photovoltaics, and an integrated bio-photovoltaic system is proposed.

[0007] The present invention discloses a conductive hydrogel, which comprises a hydrogel and a conductive material; the hydrogel comprises a gelatin-carrageenan composite hydrogel, a sodium alginate-polyvinyl alcohol composite hydrogel, a sodium alginate-polyethylene glycol composite hydrogel and a sodium alginate-carboxymethyl chitosan composite hydrogel; the conductive material comprises polypyrrole, polyaniline, carboxylated multi-walled carbon nanotubes, amino-functionalized multi-walled carbon nanotubes and conductive carbon black; the mass concentration of the conductive material is 0.1-3%.

[0008] Preferably, the mass concentration of gelatin-carrageenan in the gelatin-carrageenan composite hydrogel is 2-5%.

[0009] Preferably, the mass concentration of the conductive material is 1%.

[0010] The present invention also discloses an integrated bio-photovoltaic system, comprising: an electrochemical device; a conductive hydrogel, which is at the bottom of the chamber of the electrochemical device; genetically engineered Escherichia coli, and / or genetically engineered Shewanella and / or Geobacter, and the genetically engineered Escherichia coli, genetically engineered Shewanella and Geobacter are encapsulated in the conductive hydrogel; cyanobacteria and an algal culture medium, and the cyanobacteria and the algal culture medium are above the conductive hydrogel.

[0011] The present invention further discloses a preparation method of a conductive hydrogel, comprising the following steps: preparing a gelatin-carrageenan composite hydrogel with a mass concentration of 2-5%; adding a conductive material with a mass concentration of 0.1-3%; the conductive material is polypyrrole, polyaniline, carboxylated multi-walled carbon nanotubes, amino-functionalized multi-walled carbon nanotubes or conductive carbon black.

[0012] The present invention further also discloses a preparation method of an integrated bio-photovoltaic system, comprising the following steps: preparing a gelatin-carrageenan composite hydrogel with a mass concentration of 2-5%; adding a conductive material with a mass concentration of 0.1-3%; the conductive material is polypyrrole, polyaniline, carboxylated multi-walled carbon nanotubes, amino-functionalized multi-walled carbon nanotubes or conductive carbon black; encapsulating genetically engineered Escherichia coli, and / or genetically engineered Shewanella and / or Geobacter; adding cyanobacteria and an algal culture medium above the conductive hydrogel.

[0013] In a specific embodiment of the present invention, the components of the algal culture medium are: 17.1 g / L Na2HPO4·12H2O, 3.0 g / L KH2PO4, 8.766 g / L NaCl, 1.5 g / L NaNO3, 0.075 g / L MgSO4·7H2O, 0.027 g / L CaCl2, 0.006 g / L citric acid, 0.001 g / L EDTA-2Na, and 1 mL / L trace element stock solution.

[0014] In a specific embodiment of the present invention, the components of the trace element stock solution are: 2.86 g / L H3BO3, 1.81 g / L MnCl2·4H2O, 0.222 g / L ZnSO4·7H2O, 0.079 g / L CuSO4·5H2O, 0.391 g / L Na2MoO4·2H2O, and 0.04 g / L CoCl2·6H2O.

[0015] Advantages of the present invention:

[0016] (1) By developing a conductive hydrogel and heterotrophic microorganisms lacking aerobic respiration, the present invention successfully solves the problem of "oxygen robbing electrons" in synthetic microbiome bio-photovoltaics, realizes the assembly of photosynthetic microorganisms and heterotrophic microorganisms in the same chamber and has the function of photoelectric conversion, and develops an integrated bio-photovoltaic system.

[0017] (2) The conductive hydrogel developed by the present invention has an oxygen isolation function. Due to the large mass transfer resistance of gas molecules in the solid-liquid two-phase, the oxygen produced by cyanobacteria is not easily penetrated into the conductive hydrogel, providing an anaerobic environment for the heterotrophic microorganisms encapsulated in the conductive hydrogel.

[0018] (3) The conductive hydrogel developed by the present invention has the function of permeating energy carriers. The conductive hydrogel has solute permeability, and the energy carriers produced by cyanobacteria can diffuse into the conductive hydrogel for heterotrophic microorganisms to oxidize and generate current.

[0019] (4) The conductive hydrogel developed by the present invention has a conductive function. The conductive material is distributed in the hydrogel to form a conductive network, which can efficiently receive the electrons released by heterotrophic microorganisms.

[0020] (5) The heterotrophic microorganisms developed by the present invention cannot perform aerobic respiration, so there is no risk of energy dissipation due to aerobic respiration for the heterotrophic microorganisms escaping outside the conductive hydrogel. Description of the drawings

[0021] Figure 1 It is a test result diagram of the oxygen isolation performance of the hydrogel.

[0022] Figure 2These are the SEM images of hydrogels. Among them, a is gelatin-carrageenan; b is sodium alginate-polyvinyl alcohol; c is sodium alginate-polyethylene glycol; d is sodium alginate-carboxymethyl chitosan.

[0023] Figure 3 This is the current-time curve of different conductive hydrogels encapsulating and modifying Shewanella.

[0024] Figure 4 This is the current-time curve of polypyrrole conductive hydrogel encapsulating and modifying Shewanella.

[0025] Figure 5 This is the schematic diagram of the integrated bio-photovoltaic system constructed by the present invention.

[0026] Figure 6 This is the current-time curve of the integrated bio-photovoltaic system constructed by the present invention.

[0027] Figure 7 This is the power density curve generated by three synthetic microbiomes. Specific Embodiments

[0028] The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments.

[0029] Example 1:

[0030] Preparation of Hydrogel and Evaluation of Oxygen Barrier Performance

[0031] 1) Prepare 4 kinds of hydrogels, including: gelatin-carrageenan composite hydrogel, sodium alginate-polyvinyl alcohol composite hydrogel, sodium alginate-polyethylene glycol composite hydrogel, and sodium alginate-carboxymethyl chitosan composite hydrogel. The composite hydrogel is formed by dissolving a certain amount of hydrogel components in water, sterilizing at high temperature, and then cooling. The compound mass ratio of gelatin and carrageenan is 1:1.

[0032] 2) Make the hydrogel into a sheet about 3 mm thick and sandwich it between the left and right chambers of the electrochemical device.

[0033] 3) Add an equal amount of distilled water to the left and right chambers of the electrochemical device.

[0034] 4) Purge oxygen from the left chamber by passing nitrogen, and use a portable dissolved oxygen meter to measure the change in dissolved oxygen.

[0035] 5) Place a magnetic stirrer in the right chamber and perform magnetic stirring at a speed of 500 rpm to increase dissolved oxygen.

[0036] 6) The control group has no hydrogel, and the rest of the implementation schemes are the same as above.

[0037] 7) In addition, the microstructures of the four hydrogels were characterized by scanning electron microscopy.

[0038] The results of the oxygen barrier property tests of the four hydrogels prepared in this example are shown in Table 1 and Figure 1 .

[0039] Table 1 Oxygen Barrier Property Tests of Four Hydrogels

[0040]

[0041] From Figure 1 it can be seen that the dissolved oxygen in the control group quickly rose to the maximum value, while the dissolved oxygen in the device with the hydrogel barrier increased slowly, and the highest dissolved oxygen was lower than that in the control group, indicating that the hydrogels generally have oxygen barrier properties.

[0042] The microstructures of the four hydrogels prepared in this example are shown in Figure 2 . From Figure 2 it can be seen that the interior of the hydrogel is a porous structure, but there are differences in the pore sizes of different hydrogels, and the porous structure provides space for the encapsulation of heterotrophic microorganisms.

[0043] Example 2:

[0044] Preparation of Conductive Hydrogel and Evaluation of Conductive Performance

[0045] 1) The conductive hydrogel is prepared by adding a conductive material to the gelatin-carrageenan composite hydrogel. Five conductive materials are used in this example, including polypyrrole, polyaniline, carboxylated multi-walled carbon nanotubes, amino-functionalized multi-walled carbon nanotubes, and conductive carbon black.

[0046] 2) The composite mass concentration of gelatin-carrageenan in the conductive hydrogel is 2-5%; the mass concentration of the conductive material is 0.1-3%. One of the following conductive materials is selected respectively: polypyrrole, polyaniline, carboxylated multi-walled carbon nanotubes, amino-functionalized multi-walled carbon nanotubes, and conductive carbon black; experiments are carried out according to Table 2; after the conductive material is mixed with the hydrogel components, high-temperature sterilization is carried out; the conductivity is evaluated by encapsulating and transforming Shewanella oneidensis to generate electricity.

[0047] Table 2 Conductivity Data Sheet

[0048]

[0049] 3) The transformed Shewanella oneidensis is cultured for 24 hours, and the cells are collected by centrifugation.

[0050] 4) After the conductive hydrogel is melted and naturally cooled to below 45 °C, 25 mL is taken and added to the anodic chamber of the electrochemical device.

[0051] 5) Add the centrifugally collected engineered Shewanella cells at a cell density of OD600 = 0.75 to the conductive hydrogel, mix well, and allow it to cool and solidify naturally.

[0052] The modified Shewanella is a genetically modified Shewanella engineering strain, abbreviated as S3:

[0053] (1) Strain characteristics: Unable to perform aerobic respiration under aerobic conditions.

[0054] (2) Initial strain: Shewanella oneidensis MR-1 (ATCC 700550), both wild-type and mutant strains are acceptable. The link to Shewanella oneidensis MR-1 (ATCC 700550) is: https: / / www.atcc.org / products / 700550 .

[0055] (3) Engineering sites: Inactivate three respiratory terminal oxidase-related genes in Shewanella, including: cco (SO_2361-2364), cyd (SO_3284-3286), cox (SO_4606-4609).

[0056] The gene sequence information of cco (SO_2361-2364) is shown in Sequence Listing SEQ ID NO.1.

[0057] The gene sequence information of cyd (SO_3284-3286) is shown in Sequence Listing SEQ ID NO.2.

[0058] The gene sequence information of cox (SO_4606-4609) is shown in Sequence Listing SEQ ID NO.3.

[0059] The above gene inactivation methods can include knockout inactivation, insertion inactivation, site-directed mutagenesis inactivation, RNAi interference inactivation, dCas inhibition inactivation, etc.

[0060] 6) After the conductive hydrogel solidifies, add MBG11-S medium on top.

[0061] The MBG11-S medium has the following composition (per 1 L): 17.1 g Na2HPO4·12H2O, 3.0 g KH2PO4, 8.766 g NaCl, 1.5 g NaNO3, 0.075 g MgSO4·7H2O, 0.027 g CaCl2, 0.006 g citric acid, 0.001 g EDTA-2Na, 1 mL trace element stock solution (1000×).

[0062] The composition of the trace element stock solution is as follows (1 L): 2.86 g of H3BO3, 1.81 g of MnCl2·4H2O, 0.222 g of ZnSO4·7H2O, 0.079 g of CuSO4·5H2O, 0.391 g of Na2MoO4·2H2O, 0.04 g of CoCl2·6H2O.

[0063] Add 15 mM sodium lactate as an electron donor to the above medium.

[0064] 7) Connect a resistor between the anode and the cathode, place the electrochemical device in an incubator, and monitor the generation of current.

[0065] Gels can be formed when the mass concentration of the gelatin-carrageenan blend is between 2% and 5%. The gel strength increases with the increase in concentration. However, when the concentration is too high, the hydrogel solidifies too quickly, which is not conducive to encapsulating microbial cells. When the concentration is about 3%, the hydrogel has a moderate strength and is most suitable for encapsulating cells.

[0066] Adding a conductive material endows the hydrogel with conductivity. The addition concentration of the conductive material in the range of 0.1% - 3.0% is more suitable for modifying Shewanella to generate current, and 1.0% is the most suitable. The current generated at low concentrations is low, while high concentrations will have a certain impact on the gel-forming performance of the hydrogel.

[0067] The voltages and currents generated by encapsulating modified Shewanella with different conductive hydrogels prepared in this example are shown in Table 3 and Figure 3 . From Figure 3 it can be seen that the conductive hydrogel made of polypyrrole as the conductive material has the best conductivity.

[0068] Table 3 Voltage values generated by encapsulating modified Shewanella with different conductive hydrogels

[0069]

[0070]

[0071] Example 3:

[0072] Power Generation by Encapsulating Modified Shewanella with Polypyrrole Conductive Hydrogel

[0073] 1) Prepare a conductive hydrogel composed of gelatin-carrageenan and polypyrrole, where the blending concentration of gelatin-carrageenan is 2% - 5%, preferably 3%, and the concentration of polypyrrole is 0.1% - 3%, preferably 1%.

[0074] 2) Culture the modified Shewanella for 24 hours and collect the cells by centrifugation.

[0075] 3) Melt the conductive hydrogel and let it cool naturally to below 45 °C, then take 25 mL and add it to the anode chamber of the electrochemical device.

[0076] 4) The centrifugally collected Shewanella cells after modification were added to the conductive hydrogel at a cell density of OD600 = 0.75, mixed well, and allowed to cool and solidify naturally.

[0077] The modified Shewanella is a genetically modified Shewanella engineering strain, abbreviated as S3:

[0078] (1) Strain characteristics: Unable to perform aerobic respiration under aerobic conditions.

[0079] (2) Initial strain: Shewanella oneidensis MR-1 ATCC 700550, either wild type or mutant strain is acceptable. The link to Shewanella oneidensis MR-1 (ATCC 700550): https: / / www.atcc.org / products / 700550 .

[0080] (3) Modification sites: Inactivate three genes related to respiratory terminal oxidases in Shewanella, including: cco (SO_2361-2364), cyd (SO_3284-3286), cox (SO_4606-4609).

[0081] The gene sequence information of cco (SO_2361-2364) is shown in Sequence Listing SEQ ID NO.1.

[0082] The gene sequence information of cyd (SO_3284-3286) is shown in Sequence Listing SEQ ID NO.2.

[0083] The gene sequence information of cox (SO_4606-4609) is shown in Sequence Listing SEQ ID NO.3.

[0084] The above gene inactivation methods can include knockout inactivation, insertion inactivation, site-directed mutagenesis inactivation, RNAi interference inactivation, dCas inhibition inactivation, etc.

[0085] 5) After the conductive hydrogel solidifies, add MBG11-S medium on top.

[0086] The composition of the MBG11-S medium (per 1 L) is as follows: 17.1 g of Na2HPO4·12H2O, 3.0 g of KH2PO4, 8.766 g of NaCl, 1.5 g of NaNO3, 0.075 g of MgSO4·7H2O, 0.027 g of CaCl2, 0.006 g of citric acid, 0.001 g of EDTA-2Na, and 1 mL of trace element stock solution (1000×).

[0087] The composition of the trace element stock solution is as follows (1 L): 2.86 g of H3BO3, 1.81 g of MnCl2·4H2O, 0.222 g of ZnSO4·7H2O, 0.079 g of CuSO4·5H2O, 0.391 g of Na2MoO4·2H2O, and 0.04 g of CoCl2·6H2O.

[0088] Add 15 mM sodium lactate as an electron donor to the above medium.

[0089] 6) Connect a resistor between the anode and the cathode, place the electrochemical device in an incubator, and monitor the generation of current.

[0090] 7) The control groups include a group without a conductive hydrogel (using carbon cloth as the electrode), a cell-free group, and a group without an electron donor. The other implementation schemes are the same as above.

[0091] The voltages and currents generated by using the conductive hydrogel to encapsulate and transform Shewanella in Example 3 are shown in Table 4 and Figure 4 .

[0092] Table 4 Voltage values generated by using polypyrrole conductive hydrogel to encapsulate and transform Shewanella

[0093]

[0094]

[0095]

[0096] From Figure 4 it can be seen that the transformed Shewanella encapsulated by the conductive hydrogel generates a relatively high current, while the current generated by the non-hydrogel system is lower than that of the hydrogel system, indicating that the conductive hydrogel has excellent electrical conductivity, solute permeability, and biocompatibility. In addition, compared with the experimental group, the currents generated by the cell-free group and the group without an electron donor are extremely low, indicating that the current generated by the experimental group is due to the transformed Shewanella oxidizing the electron donor, excluding the contribution of the conductive hydrogel itself to the current.

[0097] Example 4: Construction of an Integrated Bio - photovoltaic System Using Conductive Hydrogel

[0098] The integrated bio-photovoltaic system constructed in this example is as Figure 5 shown.

[0099] The integrated bio-photovoltaic system is a single-chamber electrochemical device. One side of the electrochemical device is an air cathode; the bottom of the chamber of the electrochemical device is a conductive hydrogel, which encapsulates transformed Escherichia coli, transformed Shewanella, and Geobacter; the upper part of the chamber of the electrochemical device is cyanobacteria and an algal medium. The cyanobacteria are algal strains capable of photosynthesizing to produce organic substances, preferably sucrose-producing cyanobacteria.

[0100] The integrated bio-photovoltaic system is composed of a synthetic microbiome containing different microorganisms. Its basic principle is as follows: photosynthetic microorganisms, namely cyanobacteria, absorb light energy and store it in organic matter; the organic matter diffuses into the conductive hydrogel, and heterotrophic microorganisms (including engineered Escherichia coli, engineered Shewanella, and Geobacter) gradually oxidize the organic matter, releasing electrons and generating an electric current. The photosynthetic microorganisms and heterotrophic microorganisms jointly complete the efficient conversion of light energy to electrical energy in the same time and space. The specific implementation steps are as follows:

[0101] 1) Prepare a conductive hydrogel composed of gelatin-carrageenan and polypyrrole, where the compound concentration of gelatin-carrageenan is 2-5%, preferably 3%, and the concentration of polypyrrole is 0.1-3%, preferably 1%.

[0102] 2) Anaerobically culture the engineered Escherichia coli. The components of the anaerobic culture medium for the engineered Escherichia coli are as follows (1L): 2.31 g KH2PO4, 16.43 g K2HPO4·3H2O, 12 g tryptone, 24 g yeast extract, 4 mL glycerol, and 3 g glucose.

[0103] The modified Escherichia coli is a genetically modified Escherichia coli engineering strain, abbreviated as E3:

[0104] (1) Strain characteristics: Unable to perform aerobic respiration under aerobic conditions.

[0105] (2) Initial strain: Any Escherichia coli, such as Escherichia coli ATCC 8739, Escherichia coli ATCC 700926, Escherichia coli DSM 27469, Escherichia coli BW25113. Both wild-type and mutant strains are acceptable.

[0106] Link to the Escherichia coli ATCC 8739 strain:

[0107] https: / / www.atcc.org / products / 8739。

[0108] Link to the Escherichia coli ATCC 700926 strain:

[0109] https: / / www.atcc.org / products / 700926。

[0110] Link to the Escherichia coli DSM 27469 strain:

[0111] https: / / www.dsmz.de / collection / catalogue / details / culture / DSM - 27469。

[0112] The Escherichia coli BW25113 strain Link:

[0113] http: / / weidibio.com / display.asp?id=2910。

[0114] (3) Modification sites: Inactivate 4 respiratory terminal oxidase-related genes in Escherichia coli, including: cyoABCD, appBC, cydAB, ygiN.

[0115] The gene sequence information of cyoABCD is shown in Sequence Listing SEQ ID NO.4.

[0116] The gene sequence information of appBC is shown in Sequence Listing SEQ ID NO.5.

[0117] The gene sequence information of cydAB is shown in Sequence Listing SEQ ID NO.6.

[0118] The gene sequence information of ygiN is shown in Sequence Listing SEQ ID NO.7.

[0119] The above gene inactivation methods can include knockout inactivation, insertion inactivation, site mutation inactivation, RNAi interference inactivation, dCas inhibition inactivation, etc.

[0120] 3) Anaerobically culture the modified Shewanella. The components of the anaerobic culture medium for the modified Shewanella are as follows (1L): 10g NaCl, 10g tryptone, 5g yeast extract, 4.8g sodium fumarate.

[0121] The modified Shewanella is a genetically modified Shewanella engineering strain, abbreviated as S3:

[0122] (1) Strain characteristics: Unable to perform aerobic respiration under aerobic conditions.

[0123] (2) Initial strain: Shewanella oneidensis MR-1 (ATCC 700550), both wild-type and mutant strains are acceptable. The Shewanella oneidensis MR-1 (ATCC 700550) Link: https: / / www.atcc.org / products / 700550 .

[0124] (3) Modification sites: Inactivate 3 respiratory terminal oxidase-related genes in Shewanella, including: cco

[0125] (SO_2361-2364), cyd(SO_3284-3286), cox(SO_4606-4609).

[0126] The gene sequence information of cco (SO_2361-2364) is shown in Sequence Listing SEQ ID NO.1.

[0127] The gene sequence information of cyd (SO_3284-3286) is shown in Sequence Listing SEQ ID NO.2.

[0128] The gene sequence information of cox (SO_4606-4609) is shown in Sequence Listing SEQ ID NO.3.

[0129] The above gene inactivation methods can include knockout inactivation, insertion inactivation, site-directed mutagenesis inactivation, RNAi interference inactivation, dCas inhibition inactivation, etc.

[0130] 4) Anaerobically culture Geobacter sulfurreducens PCA, ATCC 51573, wild type. The components of the anaerobic medium for Geobacter are as follows (1L): 1.5 g NH4Cl, 0.78 g NaH2PO4·2H2O, 0.1 g KCl, 2.5 g NaHCO3, 2.46 g sodium acetate, 8.0 g sodium fumarate, 10 mL Wolfe vitamin stock solution, and 10 mL modified Wolfe trace element stock solution.

[0131] 5) Centrifuge and collect the cultured genetically modified Escherichia coli, genetically modified Shewanella, and Geobacter.

[0132] 6) Melt the conductive hydrogel and let it cool naturally to below 45 °C. Take 25 mL and add it to the anodic chamber of the electrochemical device.

[0133] 7) Mix the centrifuged and collected genetically modified Escherichia coli, genetically modified Shewanella, and Geobacter cells and add them to the conductive hydrogel. The addition amounts are 0.5 OD, 18.75 OD, and 18.75 OD respectively (corresponding to cell densities OD 600 which are 0.02, 0.75, and 0.75 respectively). Mix well and let it cool and solidify naturally.

[0134] The process of encapsulating the heterotrophic microorganisms: When the temperature of the conductive hydrogel drops below 45 °C, add the genetically modified Escherichia coli, genetically modified Shewanella, or Geobacter cells to the conductive hydrogel, mix well, and let it solidify naturally.

[0135] 8) After the conductive hydrogel solidifies, add algal medium, namely MBG11-S medium, to the upper layer. The composition of the medium is (1L): 17.1 g of Na2HPO4·12H2O, 3.0 g of KH2PO4, 8.766 g of NaCl, 1.5 g of NaNO3, 0.075 g of MgSO4·7H2O, 0.027 g of CaCl2, 0.006 g of citric acid, 0.001 g of EDTA-2Na, and 1 mL of trace element stock solution (1000×).

[0136] The composition of the trace element stock solution is as follows (1L): 2.86 g of H3BO3, 1.81 g of MnCl2·4H2O, 0.222 g of ZnSO4·7H2O, 0.079 g of CuSO4·5H2O, 0.391 g of Na2MoO4·2H2O, and 0.04 g of CoCl2·6H2O.

[0137] 9) Inoculate sucrose-producing cyanobacteria into the upper-layer medium.

[0138] 10) Use the conductive hydrogel as the anode and connect a resistor between the anode and the cathode.

[0139] The cathode is a commonly used cathode in bioelectrochemical systems, preferably an air cathode or a potassium ferricyanide cathode.

[0140] 11) Place the device in an illumination incubator for cultivation. Preferably, the conditions of the incubator are: light intensity of about 150 μmol·m -2 ·s -1 , temperature of 30 °C, and carbon dioxide concentration of 3%.

[0141] 12) Use a data acquisition system to monitor the current generated by the device.

[0142] The integrated bio-photovoltaic system constructed in this example can directly generate electricity using light (Table 5 and Figure 6 ).

[0143] Table 5 Voltage values generated by the integrated bio-photovoltaic system

[0144]

[0145]

[0146] From Figure 6 it can be seen that the average current generated by the integrated bio-photovoltaic system is 175 μA and it can continuously generate electricity for more than 20 days.

[0147] 13) In addition to encapsulating three heterotrophic microorganisms (engineered Escherichia coli, engineered Shewanella oneidensis, and Geobacter sulfurreducens), the conductive hydrogel can also encapsulate only two of them (such as engineered Escherichia coli and engineered Shewanella oneidensis) or one of them (such as engineered Shewanella oneidensis). After inoculating cyanobacteria outside the conductive hydrogel, an integrated bio-photovoltaic system can be formed.

[0148] 14) The current-power curves of the three integrated bio-photovoltaic systems are shown in Figure 7 . Among them, the power generated by the four-bacteria system (cyanobacteria-engineered Escherichia coli-engineered Shewanella oneidensis-Geobacter sulfurreducens) is 1700 mW / m 2 , the power generated by the three-bacteria system (cyanobacteria-engineered Escherichia coli-engineered Shewanella oneidensis) is 1200 mW / m 2 , and the power generated by the two-bacteria system (cyanobacteria-engineered Shewanella oneidensis) is 600 mW / m 2 .

[0149] Advantages of the present invention:

[0150] (1) By developing a conductive hydrogel and heterotrophic microorganisms lacking aerobic respiration, the present invention successfully solves the problem of "oxygen robbing electrons" in synthetic microbiome bio-photovoltaic, realizes the assembly of photosynthetic microorganisms and heterotrophic microorganisms in the same chamber and has the function of photoelectric conversion, and develops an integrated bio-photovoltaic system.

[0151] (2) The conductive hydrogel developed by the present invention has an oxygen isolation function. Due to the large mass transfer resistance of gas molecules in the solid-liquid two-phase, the oxygen produced by cyanobacteria is not easily penetrated into the conductive hydrogel, providing an anaerobic environment for the heterotrophic microorganisms encapsulated in the conductive hydrogel.

[0152] (3) The conductive hydrogel developed by the present invention has the function of permeating energy carriers. The conductive hydrogel has solute permeability, and the energy carriers produced by cyanobacteria can diffuse into the conductive hydrogel for heterotrophic microorganisms to oxidize and generate current.

[0153] (4) The conductive hydrogel developed by the present invention has a conductive function. The conductive material is distributed in the hydrogel to form a conductive network, which can efficiently receive the electrons released by heterotrophic microorganisms.

[0154] (5) The heterotrophic microorganisms developed by the present invention cannot perform aerobic respiration, so there is no risk of aerobic respiration dissipating energy for the heterotrophic microorganisms escaping outside the conductive hydrogel.

Claims

1. Integrated bio-photovoltaic system, characterized in that, Comprising: An electrochemical device; A conductive hydrogel at the bottom of the chamber of the electrochemical device; the conductive hydrogel comprises a hydrogel and a conductive material; The hydrogel is a gelatin-carrageenan composite hydrogel, a sodium alginate-polyvinyl alcohol composite hydrogel, a sodium alginate-polyethylene glycol composite hydrogel or a sodium alginate-carboxymethyl chitosan composite hydrogel; the conductive material is polypyrrole, polyaniline, carboxylated multi-walled carbon nanotubes, amino-functionalized multi-walled carbon nanotubes or conductive carbon black; the mass concentration of the conductive material is 0.1 to 3%; the mass content of gelatin-carrageenan in the gelatin-carrageenan composite hydrogel is 2 to 5%; Engineered Escherichia coli and / or engineered Shewanella and / or Geobacter, the engineered Escherichia coli and / or engineered Shewanella and / or Geobacter encapsulated inside the conductive hydrogel; The engineered Escherichia coli is Escherichia coli in which 4 respiratory terminal oxidase-related genes are inactivated; The engineered Shewanella is Shewanella in which 3 respiratory terminal oxidase-related genes are inactivated; Cyanobacteria and an algal medium, the cyanobacteria and the algal medium outside the conductive hydrogel.

2. The integrated bio-photovoltaic system according to claim 1, wherein The 4 respiratory terminal oxidase-related genes are cyoABCD , appBC , cydAB and ygiN; The said cyoABCD The gene sequence information is shown in Sequence Listing SEQ ID NO.4; The said appBC gene sequence information is as shown in Sequence Listing SEQ ID NO.5; The said cydAB The gene sequence information is shown in Sequence Listing SEQ ID NO.6; The ygiN gene sequence information is shown in Sequence Listing SEQ ID NO.

7.

3. The integrated bio-photovoltaic system according to claim 1, wherein The Shewanella bacterium ( Shewanella oneidensis ) is MR-1, including a wild type or any mutant strain thereof; The three respiratory terminal oxidase-related genes are cco , cyd and cox ; The said cco gene sequence information is shown in the sequence listing SEQ ID NO.1; The said cyd The gene sequence information is shown in Sequence Listing SEQ ID NO.2; The said cox The gene sequence information is shown in Sequence Listing SEQ ID NO.3 4. The integrated bio-photovoltaic system according to claim 1, characterized in that, The Escherichia coli is Escherichia coli ( Escherichia coli ), ATCC 8739, Escherichia coli ( Escherichia coli ), ATCC 700926, Escherichia coli ( Escherichia coli ), DSM 27469 or Escherichia coli ( Escherichia coli ), BW25113.

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